In order for cells to adhere and grow on a substrate, the interface between the substrate and the cell growth medium must possess an appropriate combination of physical and chemical properties. Control over the surface of substrate provides for control over cell adhesion. Advantageous properties imparted by a surface range from the complete rejection of any cell adhesion or growth, to cell adhesion without growth, to cell adhesion, growth, and differentiation. The property desired depends on the end-use of the substrate. For example, articles implanted in vivo, such as stents, catheters, and artificial organs, preferably do not induce biochemical processes that lead to scarring and/or rejection of said article. These implants may be advantageously coated with thin films that render them biocompatible. Alternatively, some applications, especially those in tissue engineering, require substrates that encourage the growth, differentiation, and proliferation of cells.
Recent studies have shown that some cells need to aggregate and interact with neighboring cells in a 3-D environment in order to activate or enhance metabolic activity, (see Kurosawa, H., Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. Journal of Bioscience and Bioengineering 2007, 103, (5), 389-398) and extracellular matrix production. Cell aggregates or cell spheroids of human mesenchymal (hMSCs) and pluripotent stem cells (PSCs) have demonstrated promise in cellular therapy and tissue engineering. These aggregates are usually formed by suspension culture techniques, where the cells are cultured under orbital shaking or magnetic stirring with special media to prevent differentiation, or using coated microcarrier technology, where cells proliferate onto small particles suspended in media (e.g., see U.S. Pat. No. 8,716,018 issued to Oh et al.).
Control over surface properties is maintained by an appropriately-designed coating. Ultrathin films of polyelectrolyte complex, or polyelectrolyte multilayers (PEMUs) are prepared by alternating exposure of the substrate to polyelectrolytes or charged particles. See Decher and Schlenoff, Eds., Multilayer Thin Films—Sequential Assembly of Nanocomposite Materials, Wiley-VCH, Weinheim (2003); Decher, Science, 277, 1232 (1997). Decher and Hong (U.S. Pat. No. 5,208,111) disclose a method for a buildup of multilayers by alternating dipping, i.e., cycling a substrate between two reservoirs containing aqueous solutions of polyelectrolytes of opposite charge, with an optional rinse step in polymer-free solution following each immersion.
The large library of materials available for this layer-by-layer assembly enables precise tailoring of surface properties for cell culture. There have been many reports on cells cultured on substrates modified with multilayers. While most multilayers allow some degree of cell attachment, spreading, and proliferation, others effectively prevent cells adhesion. Cell adhesion is a complex, dynamic process, even in the absence of specific interactions. The multitude of physical factors playing a role in cell attachment onto multilayers include surface roughness, wettability, swelling, internal ionic crosslinking, thickness, surface charge, and mechanical properties.
Previous observations show no correlation of cell attachment with one unique parameter. However, depending on the cell type some parameters may influence surface attachment more than others. A good deal of contradiction exists. For example, when it comes to the importance of surface charge, prior research concludes that cells can, or cannot, adhere to both positively and negatively charged surfaces, even though the toxicity of polycations is well documented. Such studies usually focus on the sign of the charge on the surface, but rarely determine the actual charge density.
For cell culture there is a need for a simple, rugged surface coating that induces cells to form clusters.